Maize (Zea mays L.) plants carrying the Texas male-sterile cytoplasm (cms-T) are particularly susceptible to the fungal pathogen Dipolaris (Helminthosporium) maydis, race T, the causative agent of Southern Corn Leaf Blight (Hooker, A. L. et al. (1970) Plant Dis. Rep. 54:708). A host-specific pathotoxin (BmT-toxin) isolated from the fungus specifically alters membrane permeabilities of mitochondria from cms-T maize. For structure of BmT and related toxins, see Frantzen, K. A. (1987) Plant Physiol. 83:863. The carbamate insecticide, methomyl, although structurally unrelated, mimics the BmT-toxin effects (Humaydan, H. S. and Scott, E. W. (1977) Hortic. Sci. 12:312). For structure of methomyl and analogs toxic to cms-T maize mitochondria, see Aranda et al. (1987) Phytochem. 26:1909. Mitochondria from maize plants containing either the S or C male-sterile or normal (male-fertile) cytoplasms are unaffected by the BmT-toxin or methomyl. The site of toxin and methomyl action is believed to be at the inner mitochondrial membrane. In response to BmT-toxin or methomyl, cms-T mitochondria exhibit rapid swelling, uncoupling of oxidative phosphorylation, inhibition of malate-driven respiration and leakage of small molecules such as NAD.sup.+ and Ca.sup.++ (Miller, R. J. and Koeppe, D. E. (1971) Science 173:67; Koeppe, D. E. et al. (1978) 201:1227; Berville, A. et al. (1984) Plant Physiol. 76:508; Klein, R. R. and Koeppe, D. E. (1985) Plant Physiol. 77:912; Holden, M. J. and Sze, H. (1984) Plant Physiol. 75:235).
A strict correlation exists between susceptibility to the B. maydis pathotoxin and the cytoplasmic male-sterility (cms) trait in maize plants containing the T cytoplasm. Both traits are maternally inherited and attempts to separate the two effects have been unsuccessful. Regeneration of cms-T maize callus from tissue cultures both with and without BmT-toxin selection has given rise to revertant plants that are not only resistant to the BmT-toxin, but are also male fertile; no stable revertants have been obtained that are male sterile and toxin resistant or male fertile and toxin sensitive (Brettell, R. I. S. et al. (1979) Maydica 24:203; Umbeck, P. F. and Gengenbach, B. G. (1983) Crop Sci. 23:584).
A possible explanation for the simultaneous reversion of the two traits is that a single locus of extranuclear origin encodes both phenotypic alternatives, i.e. male fertility and toxin resistance versus male sterility and susceptibility.
A mitochondrial gene unique to T-cytoplasm of maize has been isolated and characterized (Dewey, R. F. et al. (1986) Cell 44:439). Designated urf13-T, it encodes a 13 kd protein associated with the cms trait. The nuclear fertility restorer gene Rf1 alters the transcript of urf13-T, resulting in a significant decrease in abundance of the 13 kd protein. Also, in cms-T plants that have reverted to male fertility and B. maydis resistance, the urf13-T reading frame has been found to be either altered or the gene completely deleted. The urf13-T gene has been sequenced and the amino acid sequence of the protein it encodes has been deduced (Dewey, R. E. et al. (1987) Proc. Natl. Acad. Sci. USA 84:5374).
Preincubation of cms-T maize mitochondria with dicyclohexylcarbodiimide (DCCD), a reagent that preferentially binds covalently to carboxyl groups in hydrophobic regions of proteins, confers protection against the effects of BmT-toxin (Bouthyette, P-Y. et al. (1985) J. Exp. Bot. 36:511; Holden, M. J. and Sze, H. (1987) in Plant Mitochondria, Structural, Functional and Physiological Aspects, A. L. Moore and R. B. Beechey, (eds.) Plenum Press, New York, pp. 305-308). Pretreatment of mitochondria with 4 through 15 .mu.M DCCD prevents toxin-induced inhibition of malate-dependent oxidation, dissipation of the membrane potential, and leakage of accumulated calcium. Preincubation with the water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, does not protect cms-T mitochondria from toxin action, suggesting that DCCD modifies a component situated in a hydrophobic environment.